The present invention relates to a laser scan confocal microscope.
For example, in a confocal microscope disclosed in Japanese Patent No. 3365884 (Patent Document 1), illumination light is collected onto a sample such as a living sample, a light flux (fluorescent light) emitted from a focal portion on the sample is collected onto a confocal diaphragm plane, and a light quantity of the light flux having passed through the confocal diaphragm is detected by a photodetector. In order to obtain a two-dimensional image of the sample, the illumination light is converted into scan light by a galvano-mirror type scanner or the like, and the two-dimensional image of the sample is obtained while the sample is scanned with a focal portion (spot).
A pinhole member is disposed in the confocal diaphragm plane. Because the pinhole member transmits only a light beam collected in a pinhole (aperture) while cutting off other light beams, only the light beam emitted from a particular depth on the sample is incident to the photodetector, and the light beams emitted from other depths are not incident to the photodetector. Therefore, in the confocal microscope, an observation object can be sectioned only to an image of a thin-film layer located at the particular depth on the sample. In order to change sectioning resolution (thickness of a layer to be observed), it is necessary to change an aperture diameter of the pinhole member. The sectioning resolution is lowered when the aperture diameter is increased, and the sectioning resolution is enhanced when the aperture diameter is decreased.
Because the sectioning resolution depends on an aperture diameter of the pinhole, it is convenient that the aperture diameter of the pinhole is changed. Therefore, a method in which an aperture diameter of the pinhole can be changed like an iris diaphragm of a camera, a method in which plural pinholes having different aperture diameters are prepared such that one of the pinholes can be selected in a turret manner or a sliding manner, and a method in which plural pinholes are prepared such that an optical path can be changed, are used.
Patent Document 1: Japanese Patent No. 3365884
Patent Document 2: Japanese Patent Application Laid-Open No. 2005-275199
However, when an aperture diameter of the pinhole is decreased to enhance the sectioning resolution, there is generated a limb darkening phenomenon in which the light quantity is increased in the center of the screen while decreased in a peripheral portion, the degree of which depends on the type of the objective lens. Therefore, even if the object has even brightness (intensity of generated fluorescent light), an image having uneven brightness is obtained in the imaging result.
The limb darkening is mainly generated by vignetting and a shift of the conjugate point between a light source and the pinhole, caused by chromatic aberration of magnification. The conjugate point deviation has the following meaning. In fluorescent light observation an excitation wavelength differs from a fluorescent light wavelength. Accordingly, when the peripheral portion of the screen is scanned while the chromatic aberration of magnification exists in the optical system (particularly, in objective lens), the focal position of the fluorescent light is not matched with the pinhole position due to the chromatic aberration of magnification, and the light quantity of the fluorescent light passing through the pinhole is decreased to darken the image of the peripheral portion.
In order to solve the problem of the limb darkening, for example, image processing is performed to increase the light quantity of the peripheral portion in Japanese Patent Application Laid-Open No. 2005-275199 (Patent Document 2). However, an optical correction is desired rather than the image processing.
In view of the foregoing, an object of the invention is to provide a laser scan confocal microscope which can correct the influence of the limb darkening caused by the chromatic aberration of magnification using the optical system even if the objective lens having the chromatic aberration of magnification is used.